1. Field of the Invention
The present invention relates to a novel MAPK kinase derived from a vertebrate and a DNA coding for the same. More particularly, the present invention is concerned with a MAPK kinase which is activated by a stimulus induced by TNF-xcex1 and/or by a stimulation of Fas antigen, and which in turn activates SAPK/JNK, but does not activate p38. Also, the present invention is concerned with a DNA coding for the above-mentioned MAPK kinase. By the use of the MAPK kinase and the DNA coding for the same, it has become possible to provide a method for screening a novel substance which can be used for treating or preventing diseases resulting from an excess activation or inhibition of a MAP kinase cascade, and also to provide a diagnostic reagent for such diseases. The present invention is also concerned with a replicable recombinant DNA which comprises a replicable expression vector and, operably inserted therein, the above-mentioned DNA; a cell of a microorganism or cell culture, which is transformed with the above-mentioned replicable recombinant DNA; a polypeptide of a dominant negative form of the above-mentioned MAPK kinase and a DNA coding for the same; and an antibody capable of binding specifically to the above-mentioned MAPK kinase.
2. Prior Art
MAP (mitogen-activated protein) kinase (MAPK) was first discovered in the late 1980""s as a serine/threonine kinase (Ser/Thr kinase; i.e., an enzyme capable of phosphorylating serine or threonine residues in a protein) which is activated by stimuli, such as insulin {Sturgill, T. W. et al., Biochim. Biophys. Acta, 1092: 350-357 (1991)}, various cell growth factors and tumor promoters {Nishida, E. et al., Int. Rev. Cytol., 138: 211-238 (1992)}. Studies over the past 10 years revealed that the MAP kinase is a major functional unit of an intracellular signal transduction system which mediates cell determination and functional regulation of eukaryotic cells in response to extracellular stimuli {Nishida, E. et al., Trends Biochem. Sci., 18: 128-131 (1993); Marshall, C. J., Curr. Opin. Genet. Dev., 4: 82-89 (1994); and Cobb, M. H. et al., J. Biol. Chem., 270: 14843-14846 (1995)}. Particularly, an important achievement in the field of cell biology of the 1990""s is an elucidation of a signal transduction pathway which starts from a cell growth factor receptor having tyrosine kinase activity, through an adapter molecule composed of SH2 (Src homology 2) and SH3 (Src homology 3), Ras (an oncogene product which is a GTP-binding protein) and Raf-1 (an oncogene product which is a serine/threonine kinase), and leading to the MAP kinase. The studies revealed that this signal transduction pathway is a central pathway responsible for determining cell proliferation, cell differentiation, and cell development of higher eucaryotic organisms.
A signal transduction molecule is converted into an activated form (switched xe2x80x9conxe2x80x9d) by a signal in the upstream of a signal transduction pathway, and the activated molecule returns to an inactive form (switched xe2x80x9coffxe2x80x9d) after transducing the signal to the downstream thereof. The regulatory mechanism for switching on/off the MAP kinase has an interesting feature. That is, phosphorylation of T and Y in the TEY (Thr-Glu-Tyr in 3-letter abbreviation) sequence located in the boundary region between the kinase subdomains VII and VIII is required for activating the MAP kinase. An enzyme called MAPK kinase (MAPKK) or MAPK/ERK kinase (MEK) was identified as an enzyme which catalyzes the phosphorylation (that is, activation) of these amino acid residues. MAPK kinase is a dual specificity kinase which is capable of phosphorylating both serine/threonine residue and tyrosine residue.
For activating a MAPK kinase, it is necessary to phosphorylate two serine and/or threonine residues (i.e., two serine residues, two threonine residues, or one serine residue and one threonine residue) located in the boundary region between the kinase subdomains VII and VIII, and a serine/threonine kinase responsible for this phosphorylation is designated MAPKK kinase (MAPKKK). The above-mentioned Raf-1 is one example of MAPKK kinase, and the following cascade reaction:
Rasxe2x86x92Raf-1 (i.e., MAPKKK)xe2x86x92MAPKKxe2x86x92MAPK,
is one of the major signal transduction pathways. The cascade reaction consisting of three kinase molecules,
MAPKKKxe2x86x92MAPKKxe2x86x92MAPK,
is called a MAP kinase signal cascade.
The above-mentioned signal transduction system,
Raf-1/MAPKKKxe2x86x92MAPKKxe2x86x92MAPK,
is the first identified MAP kinase signal cascade and, therefore, this system is frequently called xe2x80x9cclassical MAP kinase signal pathwayxe2x80x9d. Later studies revealed the existence of various kinases which are similar to the classical MAP kinase. One example of such a kinase is stress-activated protein kinase (SAPK). This enzyme has been identified as a kinase which is activated in response to a stimulation of a cell by chemical stresses (such as protein synthesis inhibitor) or physical stresses (such as heat shock or change in osmotic pressure) {Kyriakis, J. M., Nature, 369: 156-160 (1994)}. SAPK was later found to be identical to c-Jun N-terminal kinase (JNK), which is a kinase identified independently from and contemporaneously with SAPK, and phosphorylates the N-terminus of transcription factor Jun to increase the transcription activity thereof {Derijard, B., Cell, 76: 1025-1037 (1994)} (hereinafter, SAPK and JNK are frequently referred to as xe2x80x9cSAPK/JNKxe2x80x9d). SAPK/JNK has homology to the classical MAP kinase, and a sequence corresponding to the TEY sequence necessary for the activation of the classical MAP kinase is TPY (Thr-Pro-Tyr) in SAPK/JNK. SAPK/JNK is similar to the classical MAP kinase in that the Thr and Tyr residues in the above-mentioned sequence are phosphorylated by a sole MAPK kinase in the upstream thereof, but the major activator of SAPK/JNK is a MAPK kinase called SAPK/ERK kinase-1 (SEK1) or mitogen-activated protein kinase kinase 4 (MKK4) (hereinafter, SEK1 and MKK4 are frequently referred to as xe2x80x9cSEK1/MKK4xe2x80x9d) {Lin, A. et al., Science, 268: 286-290 (1995); Sanchez, I., Nature, 372: 794-798 (1994); and Moriguchi, T. et al., J. Biol. Chem., 270: 12969-12972 (1995)}. Therefore, with respect to the classical MAP kinase, the classical MAPK kinase functions as an activation factor in the upstream of the signal transduction pathway, and a novel MAP kinase is phosphorylated (activated) specifically by a different MAPK kinase. With respect to a MAPKK kinase in the upstream of a pathway leading to SAPK/JNK, a kinase called MEKK is known, but the existence of other kinases capable of functioning as a MAPKK kinase is not known.
In addition to SAPK/JNK mentioned above, a kinase similar to MAP kinase, which is simply called xe2x80x9cp38xe2x80x9d after its molecular weight, is also known in the art. This kinase has been identified and cloned as a protein which is tyrosine phosphorylated in an early stage after stimulating lymphocytes {Han, J. et al., Science, 265: 808-811 (1994)}. Contemporaneously with p38, a protein which binds to a cytokine-suppressive anti-inflammatory drug (CSAID; a drug for suppressing the production of anti-inflammatory cytokines in lymphocytes) has been independently identified and called CSAID binding protein (CSBP) {Lee, J. C. et al., Nature, 372: 739-746 (1994)}. At present, this protein is confirmed to be identical with p38. Further, MPK2, an independently isolated kinase which is activated by stress stimuli, is also found to be identical with p38 {Rouse, J. et al., Cell, 78: 1027-1037 (1994)} (Hereinafter, p38, CSBP and MPK2 are frequently referred to as xe2x80x9cp38xe2x80x9d). With respect to the above-mentioned TXY sequence (X is a predetermined amino acid residue) necessary for activating a MAP kinase, the amino acid residue xe2x80x9cXxe2x80x9d is xe2x80x9cGxe2x80x9d in p38, and p38 is activated as a result of the phosphorylation of the Thr and Tyr residues by a sole MAPK kinase in the upstream thereof. MKK3 and MKK6 are known as MAPK kinases which specifically activate p38 {Moriguchi, T. et al., J. Biol. Chem., 271: 26981-26988 (1996); and Cuenda, A. et al., EMBO J., 15: 4156-4164 (1996)}. The sequences of classical MAP kinase, SAPK/JNK and p38 are homologous to each other and, therefore, they constitute a superfamily. The MAPK kinases respectively specific for the above-mentioned three MAP kinases are also homologous to each other, and the MAPK kinases also constitute a superfamily in which MEK, SEK1/MKK4, MKK3, MKK6 and such are members thereof {Kyriakis, J. M. et al., J. Biol. Chem., 271: 24313-24316 (1996); and Davis, R. J., Trends Biochem. Sci., 19: 470-473 (1994)}. On the other hand, with respect to the MAPKK kinases located in the further upstream of the signal transduction pathway which are responsible for phosphorylating and activating each MAPK kinase, the homology among the MAPKK kinases is relatively low. For example, the homology among Raf, TAK1, MEKK, MLK3, Ask1 and Mos having the MAPKK kinase activity is only about 30% even within the kinase domains. This is in agreement with the role of the whole signal transduction system which is adapted to operate respective appropriate MAP kinase signal transduction pathways in response to a wide variety of stimuli.
SAPK/JNK and p38 are not activated by the growth factors which activate the classical MAP kinase, but they are activated by stresses, such as osmotic shock and heat shock, and cytokines, such as TNF-xcex1 (tumor necrosis factor-xcex1 or cachectin) and IL-1 (interleukin 1) {Kyriakis, J. M. et al., J. Biol. Chem., 271: 24313-24316 (1996); and Davis, R. J., Trends Biochem. Sci., 19: 470-473 (1994)}. Further, SAPK/JNK and p38 are activated under conditions at which cell death, such as UV radiation, and depletion of serum and/or growth factors are induced {Kyriakis, J. M. et al., J. Biol. Chem., 271: 24313-24316 (1996); and Davis, R. J., Trends Biochem. Sci., 19: 470-473 (1994)}. Unlike classical MAP kinase which is activated by a signal transmitted from a tyrosine kinase-type receptor, the characteristic feature of the signal transduction systems for SAPK/JNK and p38 is that these systems are initiated by various signals.
TNF-xcex1 mentioned above has various effects on inflammation, tissue disorder, immune response, and cell invasion into a focus, and these effects suggest the presence of a certain relationship between TNF-xcex1 and autoimmune diseases or graft-versus-host disease (GVHD) {J. Exp. Med., 166: 1280 (1987)}. Specifically, the role of TNF-xcex1 in the onset of inflammatory arthritis, such as rheumatoid arthritis, has been conceived {Lancet, 11: 244-247 (1989); and Ann. Rheumatic Dis. 51: 480-486 (1990)}. Administration of anti-TNF-xcex1 antibody to DBA/1 mouse either before or after the onset of arthritis relieves the inflammation accompanying collagenous arthritis, and it significantly lowers the degree of joint destruction {Williams, R. O. et al., Proc. Natl. Acad. Sci. USA, 89: 9784-9788 (1992)}. In addition, effectiveness of chimeric anti-TNF-xcex1 antibody for treating rheumatoid arthritis and Crohn""s disease has been confirmed clinically {Derkx, B. et al., Lancet, 342: 173-174 (1993); Elliott, M. J. et al., Lancet, 344: 1105-1110 (1994); and Elliott, M. J. et al., Lancet, 344: 1125-1127 (1994)}.
Studies on the signal transduction mechanism of TNF-xcex1 is in a progress. Two types of receptors for TNF-xcex1, namely, TNF-R1 having a molecular weight of 55 kD and TNF-R2 having a molecular weight of 75 kD are known in the art, and recently, molecules which associate with the TNF receptors have been directly cloned by using yeast two-hybrid system. Examples of cloned molecules which associate with TNF-R1 include TRADD (TNF-R1 associated death domain protein) {Hsu, H. et al., Cell, 81: 495-504 (1995)}, TRAP1, TRAP2 {Song, H. Y. et al., J. Biol. Chem., 270: 3574-3581 (1995)} and RIP {Stanger, B. Z. et al., Cell, 81: 513-523 (1995)}. Examples of cloned molecules which associate with TNF-R2 include TRAF1 and TRAF2 {Rothe, M. et al., Cell, 78: 681-692 (1994)}. While the molecules which associate with the TNF receptors are being identified, recent studies have revealed that NF-xcexaB, ceramide kinase and MAP kinase are activated by TNF-xcex1. It is reported that cell permeable derivatives of ceramide, such as C8-Cer (N-octanoylsphingocine) and C2-Cer (N-acetylsphingocine), exhibit a function similar to that of TNF-xcex1 {Kolesnick, R. et al., Cell, 77: 325-328 (1994)}, and these substances also activate the MAP kinase and the ceramide kinase. As apparent from the above, the knowledge on the signal transduction mechanism of TNF-xcex1 is making a rapid progress, but many problems remain unsolved. For example, the relationship between a receptor-associated protein (such as TRADD) and kinases in the mechanism for activating a signal transduction system, and a MAP kinase cascade activated by TNF-xcex1 are still unknown.
The above-mentioned SEK1/MKK4 is the only MAPK kinase that is known with respect to its function to phosphorylate SAPK/JNK. A stimulus induced by TNF-xcex1 will lead to the phosphorylation of SAPK/JNK, but this stimulus does not activate SEK1/MKK4. In this situation, the present inventors predicted the existence of an unidentified MAPK kinase which is activated by TNF-xcex1 and in turn, uses SAPK/JNK as a substrate and activates SAPK/JNK. Therefore, the goal of the present invention is to isolate a novel MAPK kinase gene and a protein encoded by the same which is activated by TNF-xcex1 and which phosphorylates SAPK/JNK, and to provide a method for using the novel gene and protein in the field of pharmaceutics and clinics.
The present inventors have made extensive and intensive studies with a view toward cloning a novel MAPK kinase. Particularly, the present inventors have successfully cloned a fragment of a novel MAPK kinase from the cDNA library of Xenopus oocyte, and have found that this fragment is similar to a MAPK kinase gene of Drosophila called hep. Subsequently, this novel MAPK kinase gene fragment was used as a probe to screen a mouse brain cDNA library, and a novel mouse MAPK kinase (hereinafter, frequently referred to as xe2x80x9cMKK7xe2x80x9d) gene which is structurally belonging to the MAPK kinase family was cloned. Next, by using the nucleotide sequence of the novel mouse MAPK kinase, the present inventors have found candidates for human MKK7 among the clones registered in the EST (Expressed Sequence Tag) database. Based on the human EST sequences, the present inventors have successfully cloned the whole nucleotide sequence of human MKK7 from human heart mRNA. Unlike SEK1 and MKK6 which are the MAPK kinases known in the art, the above-mentioned mouse MKK7 specifically activates SAPK and does not activate p38 or SAPK3, and it has been confirmed that MKK7 is a MAPK kinase participating in the signal transduction pathway in vivo starting from TNF-xcex1 and leading to SAPK/JNK. In addition, the present inventors have also found the possibility for MKK7 to participate in the induction of apoptotic signals by Fas antigen. The present invention has been completed, based on these novel findings.
Therefore, it is a principal object of the present invention to provide a MAPK kinase, which is defined as a substantially pure MAPK kinase derived from a vertebrate, wherein the MAPK kinase has the following characteristics:
(a) the MAPK kinase is activated by a stimulus induced by TNF-xcex1 and/or by a stimulation of Fas antigen;
(b) the MAPK kinase activates SAPK/JNK; and
(c) the MAPK kinase does not activate p38.
It is another object of the present invention to provide a DNA coding for the above-mentioned MAPK kinase.
It is another object of the present invention to provide a polypeptide which is a dominant negative form of the above-mentioned MAPK kinase, in which the polypeptide inhibits the activation of SAPK/JNK which is induced by TNF-xcex1.
Still another object of the present invention is to provide a method for screening a substance having the capability to inhibit the activation of SAPK/JNK by the above-mentioned MAPK kinase.
The foregoing and other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and the appended claims taken in connection with the accompanying sequence listing and drawings.
In the following sequences, the left end and right end of the nucleotide sequence are the 5xe2x80x2 end and the 3xe2x80x2 end, respectively; and the left end and right end of the amino acid sequence are the N-terminus and the C-terminus, respectively.
SEQ ID NO: 1 is the nucleotide sequence of MKK7 cDNA derived from human heart and the whole amino acid sequence encoded by the nucleotide sequence;
SEQ ID NO: 2 is the whole amino acid sequence of MKK7 derived from human heart;
SEQ ID NO: 3 is the nucleotide sequence of MKK7 cDNA derived from mouse brain and the whole amino acid sequence encoded by the nucleotide sequence;
SEQ ID NO: 4 is the whole amino acid sequence of MKK7 derived from mouse brain;
SEQ ID NO: 5 is the nucleotide sequence of a cDNA of an alternatively spliced form of MKK7 derived from mouse brain and the amino acid sequence encoded by the nucleotide sequence;
SEQ ID NO: 6 is the amino acid sequence of the alternatively spliced form of MKK7 derived from mouse brain;
SEQ ID NO: 7 is the nucleotide sequence of MKK7 cDNA fragment derived from Xenopus oocyte and the amino acid sequence encoded by the nucleotide sequence;
SEQ ID NO: 8 is the amino acid sequence of MKK7 fragment derived from Xenopus oocyte;
SEQ ID NO: 9 is the nucleotide sequence of a dominant negative form of MKK7 which was synthesized based on the nucleotide sequence of SEQ ID NO: 3, and the amino acid sequence encoded by the nucleotide sequence;
SEQ ID NO: 10 is the amino acid sequence of the synthesized dominant negative form of MKK7;
SEQ ID NO: 11 is the PCR primer used in 5xe2x80x2 RACE method performed in Example 2 for amplifying the 5xe2x80x2 end of human MKK7;
SEQ ID NO: 12 is the PCR primer used in 5xe2x80x2 RACE method performed in Example 2 for amplifying the 5xe2x80x2 end of human MKK7;
SEQ ID NO: 13 is the PCR primer used in 5xe2x80x2 RACE method performed in Example 2 for amplifying the 5xe2x80x2 end of human MKK7;
SEQ ID NO: 14 is the PCR primer used in 3xe2x80x2 RACE method performed in Example 2 for amplifying the 3xe2x80x2 end of human MKK7;
SEQ ID NO: 15 is the PCR primer used in 3xe2x80x2 RACE method performed in Example 2 for amplifying the 3xe2x80x2 end of human MKK7;
SEQ ID NO: 16 is the 5xe2x80x2 end primer used in Example 2 for amplifying the sequence in-between the 5xe2x80x2 and 3xe2x80x2 sequences of human MKK7;
SEQ ID NO: 17 is the 3xe2x80x2 end primer used in Example 2 for amplifying the sequence in-between the 5xe2x80x2 and 3xe2x80x2 sequences of human MKK7;
SEQ ID NO: 18 is the synthetic oligonucleotide used in Example 4 for preparing the dominant negative form of MKK7;
SEQ ID NO: 19 is the synthetic oligonucleotide used in Example 5 for preparing vector pSRxcex1-HA1; and
SEQ ID NO: 20 is the synthetic oligonucleotide used in Example 5 for preparing vector pSRxcex1-HA1.